Microphthalmia (Mi) is a Myc-related transcription factor essential for melanocyte development. Mi deficiency in humans with Waardenburg Syndrome 2A or mice produces melanocyte absence. In the current grant period we have extensively characterized Mi's DNA binding specificity that may coordinately regulate the 3 major pigment enzyme gene promoters. We found that Mi expression is profoundly regulated by Melanocyte Stimulating Hormone (MSH) via a cAMP signaling cascade whose effects on Mi's promoter are melanocyte-specific. These findings suggest that Mi participates in the differentiation pathway for pigmentation. However, Mi is also essential for melanocyte viability. Along these lines we have identified Mi as a target of c-Kit signaling in melanocytes, and elucidated a pathway in which Kit superactivates Mi via phosphorylation, recruitment of a transcriptional coactivator, and regulation of Mi stability. We also found that Mi expression is regulated by Wnt signaling via b-catenin that is aberrantly upregulated or mutated in a significant fraction of human melanomas. Mi may regulate S phase entry and mediate the mitogenic effect of b-catenin. The current proposal builds on these pigmentation and proliferation roles for Mi. The First Specific Aim will mechanistically examine Mi's biochemical modification by phosphorylations that alter transactivation potential and stability. The Second Specific Aim will focus on how the Mi promoter is regulated in a tissue-restricted fashion in melanocytes. Pathways impinging on this promoter include MSH and Wnt signaling. Promoter analyses will be done in vitro and in vivo validated using transgenic mice. The Third Specific Aim will dissect Mi's cell cycle regulatory activity both mechanistically in vitro and using mouse models that address Mi's role in melanoma proliferation. The final Specific Aim is a large effort to identify Mi's transcriptional target genes. We have initiated pilot studies together with researchers at the MIT Genome Center using oligonucleotide arrays, and will examine Mi's targets in primary melanocytes, in the context of signaling pathways that utilize Mi (MSH and c-Kit). Through an understanding of the means by which Mi is regulated as well as the transcriptional program(s) it activates, Mi may reveal a great deal about melanocyte biology.